A substantial body of literature supports linkages between osteogenic and angiogenic signals both in vitro and in vivo, but little is known about how their spatial and temporal coordination impacts the differentiation of adult stromal/stem cells (ASC) and bone regeneration. This study explores the coordination of angiogenic and osteogenic signals utilizing two techniques, recently described by our labs; i) the production ASCs sheets that can be stacked to generate quasi three- dimensional (3-D) structures and ii) the photo-controlled differentiation of ASCs through tightly regulated miRNA mimic delivery. The combination of these techniques will allow the induction of spatiotemporal gradients of angiogenic and osteogenic factors in a 3-D model to study how the timing and magnitude of differentiation cues impact ASC cell fate in the complex fracture site environment. Thus, our overall goal for this project is the development of an in vitro, quasi 3-D model of ASC osteogenesis to establish the effect of spatiotemporally modulated osteogenic and angiogenic cues and correlation of these results in a nude mouse calvarial defect model. As noted above we have developed a method of ASC cell sheet production utilizing a thermally reversible methylcellulose hydrogel polymer and automated cell manipulation system enabling the development of quasi 3-D structures to serve as model tissues. We have also demonstrated a light activated Plasmonic Gene Delivery system (PGDs), which serves as a convenient inducible gene delivery vehicle. When combined with miRNA mimics, miR-148b and miR-132, PGDs have been demonstrated to induce de novo osteogenic and angiogenic differentiation in human adipose derived ASCs, respectively. Combining both these methods will allow the induction of spatiotemporal differentiation gradients within the quasi 3-D ASC sheets and improve our understanding of the interplay of osteogenic and angiogenic factors in ASC based bone repair. The hypothesis will be tested in the following three Aims: Aim 1 will assess the impact on cell sheet stacking on ASC viability and differentiation. ASC cell sheets will be stacked into 1, 2, 5, 10 and 20 layers and cultured for up to 28 days to determine the impact on ASC viability, proliferation and differentiation potential. Aim 2 will assess the impact of temporally and spatialy varying the light activation of PGDs with osteogenic and angiogenic miRNA mimics on the osteogenesis and angiogenesis of ASCs within the quasi 3-D stem cells sheets. In aim 3 we will correlate in vitro results with in vivo in a 12-week nude mouse calvarial defect model. Optimal 3-D stack sizes and spatiotemporal induction conditions determined in Aim 1&2 will be tested in the repair of a calvarial defect in a CD-1 nude mouse. Animals will be euthanized at 1,6 and 12 weeks post-surgery and undergo histological, x-ray and ?CT evaluation for bone formation and vascularization of the defect site. The successful conduct of the project will provide a greater understanding of how spatiotemporally coordinated expression of osteo- and angiogenic factors impact ASC differentiation into bone and additionally, this research will lead to improved stromal/stem cell based therapies for critical sized bone defect repair.